Tophat Chapter 15: Enzyme Regulation

What are the factors that affect enzymatic activity?

1. Temperature: all enzymes work most efficiently at an optimum temperature

2. pH: all enzmes have an optimum pH at which they work most efficinetly

3. Presence of inhibitor: inhibitors reduce (or have no effect on) enzymatic activity

4. Substrate concentration: as an enzyme becomes saturated with substrate, the rate of reaction stays constant.

5. Enzyme concentration: more the concentration of enzyme, more is the rate of reaction.

6. Presence of activator: activators increase enzymatic activity.

What is enzyme regulation and what is its importance.

Enzyme regulation refers to the mechanisms that control enzyme activity and ensure that metabolic processes are properly coordinated. The importance is that it is necessary for maintaining homeostasis, controlling metabolic pathways, and responding to environmental changes.

What factors affect regulation?

• The availability of substrates and cofactors usually determines how fast the reaction goes

• As product accumulates, decrease rate of the enzymatic reaction

• Genetic regulation of enzyme synthesis and decay determines the amount of enzyme present at any moment.

• Allosteric regulator or inhibitor presence

• Zymogens, isozymes, and modulator proteins.

What are the different types of enzyme regulation?

• Allosteric regulation

• Proteolytic activation

• Covalent modification

• Genetic control of enzymes

• Feedback inhibition

What is cooperativity

The interaction between different subunits of an enzyme or protein in response to ligand binding, which affects the binding affinity of other subunits.

What curve does cooperativity create?

Sigmoidal (S-shaped)

What does positive cooperativity do?

Amplifies enzyme activity by increasing the affinity of S to E once the first molecule binds, making it easier for additional molecules to bind.

What does negative cooperativity do?

Dampens enzyme activity by reducing affinity of S to E, decreasing the likelihood of further binding after the first molecule attaches. Flattens the binding curve, resulting in a slower increase in binding or reaction rate as ligand concentration increases. Reduces the risk of over-activation by moderating the response as more ligand is present.

What are isozymes?

Enzymes with sightly different subunits

What is allosteric regulation?

The regulation of enzyme activity through binding of an effector molecule at a site other than the active site (allosteric site)

What are the two types of effectors with allosteric regulation?

1. Feed-forward (positive)

2. Feedback (negative)

What kind of curve does allosteric regulation form?

Sigmoidal (S-shaped)

What are the two models of allosteric regulation?

MWC and KNF

What are the components of the Monod, Wyman, and Changeux symmetric model (MWC)?

• Different conformations have different affinities for the various ligands.

• All the subunits of an oligomer must be in the same state

What are the two states for allosteric proteins?

R states (relaxed) and T states (taut)

How is positive cooperativity achieved in the MWC model?

S binding increases the population of R, which increases the sites available to S.

What are heterotropic effectors?

Molecules that influence the binding of something other than themselves

What are homotropic effectors?

Molecules that influence the binding of themselves.

What are the effects of the inhibitors in the symmetry model?

• I increases the number of T conformers

• I inhibits association of S and A with R

• Saturation curve becomes more sigmoidal

What are the effects of the activators in the symmetry model?

• A increases the number of R conformers

• More binding sites for S

• Saturation curve becomes more hyperbolic

What is the Koshland, Nemethy, and Fimer ((KNF) sequential model?

Ligand binding triggers a conformation change in a protein. The KNF model explains how ligand-induced conformation changes could cause subunits to adopt conformations with little affinity for the ligand- negative cooperativity. The ligand-induced conformational change in one subunit can affect the adjoining subunit.

What are the components of proteolytic activation?

• Some enzymes are synthesized as inactive precursors (zymogens) and require proteolytic cleavage for activation.

• Irreversibility: once cleaved, zymogens cannot return to the inactive state

• This mechanism ensures that enzymes are activated only when and where they are needed, preventing premature enzyme activity that could be harmful to cells or tissues.

• Mainly used for digestive enzymes

What are the components of covalent modification?

• Enzyme activity is regulated through the addition or removal of chemical groups. (phosphorylation, methylation, acetylation)

• Reversible modifications

• Each protein kinase targets specific proteins for phosphorylation

• Phosphoprotein phosphatases catalyze the reverse reaction- removing phosphoryl groups from proteins. W

What is a reversible modification?

Phosphorylation/ dephosphorylation by kinases and phosphatases

How are kinases often regulated?

Intrasteric control in which a regulatory subunit has a pseudosubstrate sequence that mimics the target sequence, minus the phosphorylatable residue.

What residues do protein kinases phosphorylate?

Ser, Thr, and Tyr

What is cAMP

cyclic AMP dependent protein kinase. A 170-kD R2C2 tetramer in mammalian cells. Two R (regulatory) subunits bind two equivalents of cAMP each; cAMP binding releases the R subunits from the C (catalytic subunits). C subunits are enzymatically active as monomers

What is the structure of glycogen phosphorylase (GP)

• GP is a dimer of identical 842 residue subunits

• Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface

• a regulatory phosphorylation site is located at Ser14 on each subunit

• Aln allosteric effector site exerts regulatory control

• Each subunit contributes a '“tower helix” (residues 262 to 278) to the subunit-subunit interface.

• In the dimer the tower helices extend from their respective subunits and pack against each other

• Muscle GP is a dimer of identical subunits, each with PLP covalently linked

How is GP controlled?

By allosteric regulation and covalent modification

What model does GP conform to?

The MWC model

How does GP conform to the MWC model?

• The active form of the enzyme is designated to the R state

• The inactive form of the enzyme is denoted the T state

• AMP promotes the conversion of T to R

• ATP glucose 6-P and caffeine favor the conversion of R to T

• A significant conformation change occurs at the subunit interface between T and R state

• The enzyme is allosterically activated by AMP and inhibited by ATP and glucose-6-P

How is cAMP a second messenger?

It is the intracellular agent of extracellular hormones.

What is transcriptional control?

Gene expression can be upregulated or downregulated in response to cellular needs

What is feedback inhibition and its mechanism?

A product of metabolic pathway inhibits an earlier step in the pathway to regulate the flux of intermediates. Mechanism: prevents overproduction of end products.